David R. Maneval

MTHFR 677C→T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation

BACKGROUND The methylenetetrahydrofolate reductase (MTHFR) genotype is associated with modification of disease and risk of neural tube defects. Plasma and red blood cell (RBC) folate and plasma homocysteine concentrations change in response to daily intakes of folic acid supplements, but no large-scale or population-based randomized trials have examined whether the MTHFR genotype modifies the observed response. OBJECTIVE We sought to determine whether the MTHFR 677C→T genotype modifies the response to folic acid supplementation during and 3 mo after discontinuation of supplementation. DESIGN Northern Chinese women of childbearing age were enrolled in a 6-mo supplementation trial of different folic acid doses: 100, 400, and 4000 μg/d and 4000 μg/wk. Plasma and RBC folate and plasma homocysteine concentrations were measured at baseline; after 1, 3, and 6 mo of supplementation; and 3 mo after discontinuation of supplementation. MTHFR genotyping was performed to identify a C→T mutation at position 677 (n = 932). RESULTS Plasma and RBC folate and homocysteine concentrations were associated with MTHFR genotype throughout the supplementation trial, regardless of folic acid dose. MTHFR TT was associated with lower folate concentrations, and the trend of TT < CC was maintained at even the highest doses. Folic acid doses of 100 μg/d or 4000 μg/wk did not reduce high homocysteine concentrations in those with the MTHFR TT genotype. CONCLUSION MTHFR genotype was an independent predictor of plasma and RBC folate and plasma homocysteine concentrations and did not have a significant interaction with folic acid dose during supplementation. This trial was registered at clinicaltrials.gov as NCT00207558.

B vitamin intakes and incidence of colorectal cancer: results from the Women's Health Initiative Observational Study cohort.

BACKGROUND The role of one-carbon metabolism nutrients in colorectal carcinogenesis is not fully understood. Associations might be modified by mandated folic acid (FA) fortification or alcohol intake. OBJECTIVE We investigated associations between intakes of folate, riboflavin, vitamin B-6, and vitamin B-12 and colorectal cancer (CRC) in the Womens Health Initiative Observational Study, stratified by time exposed to FA fortification and alcohol intake. DESIGN A total of 88,045 postmenopausal women were recruited during 1993-1998; 1003 incident CRC cases were ascertained as of 2009. Quartiles of dietary intakes were compared; HRs and 95% CIs were estimated by Cox proportional hazards models. RESULTS Dietary and total intakes of vitamin B-6 in quartile 4 compared with quartile 1 (HR: 0.80; 95% CI: 0.66, 0.97 and HR: 0.80; 95% CI: 0.66, 0.99, respectively) and total intakes of riboflavin (HR: 0.81; 95% CI: 0.66, 0.99) were associated with reduced risk of CRC overall and of regionally spread disease. In current drinkers who consumed <1 drink (13 g alcohol)/wk, B vitamin intakes were inversely associated with CRC risk (P-interaction < 0.05). Dietary folate intake was positively associated with CRC risk among women who had experienced the initiation of FA fortification for 3 to <9 y (P-interaction < 0.01). CONCLUSIONS Vitamin B-6 and riboflavin intakes from diet and supplements were associated with a decreased risk of CRC in postmenopausal women. Associations of B vitamin intake were particularly strong for regional disease and among women drinkers who consumed alcohol infrequently. Our study provides new evidence that the increased folate intake during the early postfortification period may have been associated with a transient increase in CRC risk.

Daily intake of 4 to 7 μg dietary vitamin B-12 is associated with steady concentrations of vitamin B-12–related biomarkers in a healthy young population

BACKGROUND Studies have questioned whether the current Recommended Dietary Allowance (RDA) of 2.4 microg vitamin B-12/d is adequate. OBJECTIVE We examined the association between dietary vitamin B-12 intake and biomarkers of vitamin B-12 status. DESIGN Dietary vitamin B-12 intake was estimated, and biomarkers of vitamin B-12 status were measured, in healthy men and women (n = 299; age range: 18-50 y) who were recruited from a Florida community. The National Cancer Institute Diet History Questionnaire was used. Plasma cobalamin, total transcobalamin, holo-transcobalamin, methylmalonic acid (MMA), total homocysteine (tHcy), and autoantibodies against intrinsic factor (IF) and Helicobacter pylori were analyzed in blood samples. RESULTS Antibodies to H. pylori were detected in 12% of subjects (35/299), and negative results for IF antibodies were obtained for all subjects. The intake of vitamin B-12 correlated significantly with cobalamin, holo-transcobalamin, MMA, and tHcy. Subjects were divided into quintiles on the basis of their dietary vitamin B-12 intake (range: 0.42-22.7 microg/d), and biomarkers of vitamin B-12 status were plotted against estimated dietary vitamin B-12 intake. All biomarkers appeared to level off at a daily dietary vitamin B-12 intake between 4.2 and 7.0 microg. CONCLUSION In persons with normal absorption, our data indicate that an intake of 4-7 microg vitamin B-12/d is associated with an adequate vitamin B-12 status, which suggests that the current RDA of 2.4 microg vitamin B-12/d might be inadequate for optimal biomarker status even in a healthy population between 18 and 50 y of age.

Genomic DNA Methylation Changes in Response to Folic Acid Supplementation in a Population-Based Intervention Study among Women of Reproductive Age

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (−14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Womens Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.

Folate-mediated one-carbon metabolism genes and interactions with nutritional factors on colorectal cancer risk: Women's Health Initiative Observational Study

Investigations of folate‐mediated one‐carbon metabolism (FOCM) genes and gene‐nutrient interactions with respect to colorectal cancer (CRC) risk are limited to candidate polymorphisms and dietary folate. This study comprehensively investigated associations between genetic variants in FOCM and CRC risk and whether the FOCM nutrient status modified these associations.

Contribution of seafood to total vitamin B12 intake and status of young adult men and women.

Previous research suggests 20% of omnivores and 40% of vegetarians do not consume enough vitamin B12. The contribution of seafood to dietary B12 intake stratified by frequency of seafood consumption and B12 status of young adults was assessed. Seafood was the greatest contributor to B12 intake (31%) among nonvegetarians, and intake comparisons based on frequency of seafood consumption revealed that frequent seafood consumers had higher (p < 0.001) B12 intakes. None of the frequent seafood consumers had suboptimal status, which occurred mainly in subjects consuming seafood < 1 time/month and vegetarians. Our findings suggest that modest seafood intake contributes to maintaining normal B12 status.

Folate-mediated one-carbon metabolism genes and interactions with nutritional factors on colorectal cancer risk

Investigations of folate‐mediated one‐carbon metabolism (FOCM) genes and gene‐nutrient interactions with respect to colorectal cancer (CRC) risk are limited to candidate polymorphisms and dietary folate. This study comprehensively investigated associations between genetic variants in FOCM and CRC risk and whether the FOCM nutrient status modified these associations.

Folate-mediated one-carbon metabolism genes and interactions with nutritional factors on colorectal cancer risk: Women's Health Initiative Observational Study: One-Carbon Gene-Nutrient Interactions

Investigations of folate‐mediated one‐carbon metabolism (FOCM) genes and gene‐nutrient interactions with respect to colorectal cancer (CRC) risk are limited to candidate polymorphisms and dietary folate. This study comprehensively investigated associations between genetic variants in FOCM and CRC risk and whether the FOCM nutrient status modified these associations.

Hypomethylation of Serum Blood Clot DNA, but Not Plasma EDTA-Blood Cell Pellet DNA, from Vitamin B12-Deficient Subjects

Vitamin B12, a co-factor in methyl-group transfer, is important in maintaining DNA (deoxycytidine) methylation. Using two independent assays we examined the effect of vitamin B12-deficiency (plasma vitamin B12<148 pmol/L) on DNA methylation in women of childbearing age. Coagulated blood clot DNA from vitamin B12-deficient women had significantly (p<0.001) lower percentage deoxycytidine methylation (3.23±0.66%; n = 248) and greater [3 H]methyl-acceptance (42,859±9,699 cpm; n = 17) than DNA from B12-replete women (4.44±0.18%; n = 128 and 26,049±2,814 cpm; n = 11) [correlation between assays: r = –0.8538; p<0.001; n = 28]. In contrast, uncoagulated EDTA-blood cell pellet DNA from vitamin B12-deficient and B12-replete women exhibited similar percentage methylation (4.45±0.15%; n = 77 vs. 4.47±0.15%; n = 47) and [3 H]methyl-acceptance (27,378±4,094 cpm; n = 17 vs. 26,610±2,292 cpm; n = 11). Therefore, in simultaneously collected paired blood samples, vitamin B12-deficiency was associated with decreased DNA methylation only in coagulated samples. These findings highlight the importance of sample collection methods in epigenetic studies, and the potential impact biological processes can have on DNA methylation during collection.